Interpretive Summary: Insect-killing nematodes are small round worms that kill insect pests but don't harm people or the environment. A wide variety of insect pests can be controlled with these nematodes such as various weevils, fungus gnats, fleas, and caterpillars. Some kinds of nematodes are better at killing insects than others. Therefore, it is sometimes necessary to preserve the nematodes long-term so that the ones of most use are not lost. One way of preserving nematodes is by freezing them in liquid nitrogen. In this study we discovered how to improve the process of nematode storage in liquid nitrogen. We found that increasing the number of nematodes that are stored together increases the percentage that survives. With this approach we achieved 100% survival following storage in liquid nitrogen.

Technical Abstract: Entomopathogenic nematodes (EPNs) are used for biological control of insect pests. A theory for improved cryopreservation of nematodes has been discovered. The survival rate for infective juveniles (IJs) after cryopreservation not only relies on the concentration of glycerol (a cryoprotectant) solution, but is also correlated with the concentrations of IJs in diluted glycerol solution before storage, and in Ringer's solution after the storage. Following the guidance of this theory, a relatively high IJ concentration during cryopreservation of Steinernema carpocapsae resulted in 100% of survival and 100% retention of original virulence to Galleria mellonella larvae. This is the first report for achieving 100% survival of EPNs after preservation in liquid nitrogen. The theory was also proved for preservation of Heterorhabditis bacteriophora.